Fig 1.
Increased resistance in PI3Kγ-/- mice infected with PbA.
Percentage of survival in PbA-infected mice (A). C57BL/6 WT (solid lines, n = 5) and PI3Kγ-/- mice (dashed lines, n = 6). The survival curve of PI3Kγ-/- was significantly different of WT, with *p<0.05. Percentage of ECM incidence in infected mice (B) based on typical neurological symptoms of ECM, with *p<0.05 between groups. Parasitemia levels (C) from day 3 p.i. up to day 12 p.i., without differences between infected groups. Parasitemia was expressed as mean ± SD. Representative results from 2 independent experiments.
Fig 2.
Infected PI3Kγ-/- mice present mild histopathological changes in brain.
Representative photomicrographs of hematoxylin/eosin (HE)-stained brain sections from uninfected control mice (A, D), or PbA-infected C57BL/6 (B, E) and PI3Kγ-/- (C, F) mice on day 6 p.i. Normal histological appearance of cerebrum (A) and brainstem (D) from uninfected control mice. Brain sections from a PbA-infected wild-type animal exhibiting intravascular leukocyte aggregates (asterisks) in cerebrum (B) and hemorrhage (arrow head) in brainstem (E). Note vascular plugging (asterisk) in the deep cerebral cortex (C) and small hemorrhagic foci (arrow head) in the brainstem (F) from PbA-infected PI3Kγ-/- animal. The severity of microvascular obstruction and cerebellar hemorrhagic areas (μm2/field) in infected mice demonstrated a significant reduction in brain alterations in PI3Kγ-/- mice (J). Immunohistochemical staining for anti-Iba-1 showing no reactive microglia (arrow) in non-infected WT mouse brain (G). Microglial cells (arrows) displaying morphological changes (thickening of the microglial processes) and immunoreactivity for Iba-1 in the cerebrum of PbA-infected wild-type animal (H) and PI3Kγ-/- mouse (I). Quantification of reactive microglia (K) confirmed lower number in brain sections of infected-PI3Kγ-/- mice when compared with PbA-infected WT animal. Significant differences were indicated by *p<0.05 (n = 5 mice per group). Original magnification: A-I: x200. Sections were captured with a digital camera (Optronics DEI-470) connected to a microscope (Olympus IX70).
Fig 3.
Reduced CD8+ T cell accumulation, CD8+ T-cell cytotoxicity and activation in the brain of PbA-infected PI3Kγ-/- mice.
Flow cytometric analyses of brain-sequestered leukocytes from PbA-infected WT and PI3Kγ-/- mice on day 6 p.i. Absolute numbers of CD3+CD8+ T cells, expressed as total number per brain (A). Percentage of Granzyme B+ (GrzB+) cells in CD8+ T population (B) and the expression of CD44 in CD8+ T cells (C) presented as mean fluorescence intensity (MFI). Results are expressed as mean ± SD and are representative of 2 independent experiments (n = 4 samples per group, with each sample representing a pool of 2 mice brains). Significant difference between infected groups was indicated by *p<0.05 or **p<0.01.
Fig 4.
Reduced protein levels of phospho-IκB-α and Granzyme B in brain extracts of infected PI3Kγ-/- mice.
Western Blot analysis of phospho-IκB-α (40kDa) and Granzyme B (30kDa) in whole brain extracts, as a marker of NF-κB activation and T cell cytotoxicity, respectively. Samples from WT and PI3Kγ-/- mice (n = 2 for non-infected mice and n = 5 for PbA-infected mice, day 6 p.i.) were collected and processed for protein extraction. Brain extracts (40g) were fractioned on 10% SDS-PAGE, transferred onto nitrocellulose membrane, and then probed with anti-phospho-IκB-α, Granzyme B or anti--actin antibody, for loading control (A). Densitometric analysis are presented as arbitrary units (B) and revealed marked increase of phospho-IκB-α and Granzyme B at day 6 p.i., in infected WT mice. The expression of these two proteins was significantly lower in infected PI3Kγ-/- mice when compared with infected WT mice. Results are expressed as mean ± SEM and are representative of 2 independent experiments. Significant differences were indicated by *p<0.05 or **p<0.01.
Fig 5.
Lung pathology induced by PbA infection seems to be independent of PI3Kγ.
Representative photomicrographs of hematoxylin/eosin (HE)-stained lung sections from uninfected control mice (A), or PbA-infected C57BL/6 (B) and PI3Kγ-/- (C) mice on day 6 p.i. Normal histological section of lung parenchyma with standard architecture from uninfected control (A). Lung sections from a PbA-infected C57BL/6 and PI3Kγ-/- animal exhibiting slight interstitial edema, thickening of alveolar septae and a reduction of alveolar size (B, C). Semi-quantification score for histopathological changes in the lung of PbA-infected mice showed no significant difference between C57BL/6 and PI3Kγ-/- infected mice (n = 5 mice for each group) (D). Original magnification: A-D: x200. Sections were captured with a digital camera (Optronics DEI-470) connected to a microscope (Olympus IX70).
Fig 6.
Treatment with a PI3Kγ inhibitor delayed mortality in WT PbA-infected mice.
The selective inhibitor of PI3Kγ, AS605240, was given orally, once a day, at the dose of 30 mg/kg, from day 3 until day 6 p.i. (n = 5). Untreated infected animals (n = 8) received drug vehicle in the same therapeutic scheme. Percentage of survival in PbA-infected mice (A), where solid lines denote vehicle-treated mice and dashed lines, AS605240-treated mice. Parasitemia levels on day 6 p.i., without differences between infected groups (B). Significant differences were indicated by *p<0.05 and parasitemia expressed as mean ± SD.